Chemokines are secreted pro-inflammatory proteins of small dimensions (70-130 amino acids) mostly involved in the directional migration and activation of cells, especially the extravasation of leukocytes from the blood to tissue localizations needing the recruitment of these cells (Baggiolini M et al., 1997; Rossi D and Zlotnik A, 2000; Fernandez E J and Lolis E, 2002). Usually chemokines are produced at the site of an injury, inflammation, or other tissue alteration in a paracrine or autocrine fashion, triggering cell-type specific migration and activation.
Depending on the number and the position of the conserved cysteines in the sequence, chemokines are classified into C-, CC-, CXC- and CX3C-chemokines. Inside each of these families, chemokines can be further grouped according to the homology of the entire sequence, or of specific segments.
A series of heptahelical G-protein coupled membrane receptors, are the binding partners that allow chemokines to exert their biological activity on the target cells, which present specific combinations of receptors according to their state and/or type. An unified nomenclature for chemokine ligands and receptors, which were originally named by the scientists discovering them in a very heterogeneous manner, has been proposed to associate each of these molecule to a systemic name including a progressive number CCL1, CCL2, etc. for CC-chemokines; CCR1, CCR2, etc. for CC-chemokines receptors, and so on.
The physiological effects of chemokines result from a complex and integrated system of concurrent interactions. The receptors often have overlapping ligand specificity, so that a single receptor can bind different chemokines, as well a single chemokine can bind different receptors. In particular, N-terminal domain of chemokines is involved in receptor binding and N-terminal processing can either activate chemokines or render chemokines completely inactive.
Amongst all the chemokines characterized so far, CC-chemokines, such as CCL5 (also known as RANTES; Appay V and Rowland-Jones S L, 2001) or CCL3 (also known as MIP-1alpha, U.S. Pat. No. 6,355,476), have been intensively studied to identify therapeutically useful molecules. Variants of CC-chemokines, missing up to nine N-terminal amino acids, have been tested for their activity as inhibitors or antagonists of the naturally occurring forms. These molecules are inactive on monocytes and are useful as receptor antagonists (Gong J H et al., 1996; WO 99/16877). Alternatively, N-terminal extension of the mature CC-chemokine with one Methionine results in almost complete inactivation of the molecule, which also behaves as an antagonist for the authentic one (WO 96/17935).
Moreover, in order to perform structure-function analysis of CC-chemokines, variants containing substitutions or chemical modifications in different internal positions, as well as CC-chemokine derived peptides, have been tested for the interactions with receptors or other molecules. Some of these variants have been disclosed as having significatively altered binding properties, and sometimes they are active as CC-chemokine antagonists, having potential therapeutic applications in the treatment of HIV infection and some inflammatory or allergic diseases (WO 99/33989; Nardese V et al., 2001). In particular, the binding determinants and the physiological relevance of the interactions of chemokines, by the means of specifically positioned basic residues, with Glycosaminoglycans (GAGs) has been intensively studied (WO 02/28419; Vives R et al., 2002; McCornack M A et al., 2003; Stringer S E et al., 2002; Fukui S et al., 2002; Laurence J S et al., 2001; Martin L et al., 2001; Koopmann W and Krangel M S, 1997).
Even though there are potential drawbacks in using chemokines as therapeutic agents (tendency to aggregate, promiscuous binding), these molecules offer the possibility for therapeutic intervention in pathological conditions associated to such processes, in particular by inhibiting/antagonizing specific chemokines and their receptors at the scope to preventing the excessive recruitment and activation of cells, in particular leukocytes, for a variety of indications related to inflammatory and autoimmune diseases, cancers, and bacterial or viral infections (Schneider G P et al., 2001, Baggiolini M, 2001; Godessart N and Kunkel S L, 2001).
The possible therapeutic applications of chemokine-related compounds against hepatic diseases have been intensively studied, as recently reviewed (Ajuebor M N et al., 2002; Marra F, 2002, Colletti L M, 1999). In particular, liver specific inflammation is mediated by activated CD4(+) T cells and driven by an upregulation of the hepatic expression of IFNgamma, but the mechanisms governing T cell migration from the blood into tissues during T cell-mediated hepatitis remains incompletely understood, since the endogenous mediators that promote the recruitment of T cells to the liver during T cell-mediated liver diseases have been poorly characterized.
It has been demonstrated that some chemokines are highly expressed and important for the recruitment for liver-infiltrating lymphocytes in hepatitis-related animal models (acetaminophen-induced, Concanavalin A-induced, adenovirus-induced, or hepatitis B virus-specific), suggesting a specific role of these molecules in the development of hepatitis (Bautista A P, 2002; Lalor P F et al., 2002; Hogaboam C M et al., 2000; Kusano F et al., 2000).
Some broad spectrum CC-chemokine antagonists were disclosed in connection to hepatic diseases (WO 00/73327; WO 01/58916; U.S. Pat. No. 6,495,515). CXC chemokines are capable to induce rapid hepatocyte proliferation and liver regeneration after injury (WO 01/10899). CCR1 or MIP-1alpha antagonists can be used for inhibiting graft-related or ischemia/reperfusion-related liver dysfunctions (WO 00/44365; Murai M et al., 1999).
However, prior art fails to describe any therapeutic efficacy of an isolated, specific CC-chemokine mutant generated by the substitution of internal residues, against liver fibrotic inflammatory and/or autoimmune diseases.